Nuclear isolation medium and procedure for separating cell nuclei

ABSTRACT

Nuclear isolation media and procedures are described for dissociating discrete, non-agglomerated cell nuclei from animal tissue without the need to use enzyme treatments, centrifugation or the like in order to achieve the desired separation. The media facilitates separation and maintains the nuclear membrane intact and in its normal physiological environment. When a DNA-fluorochrome stain is included in the medium an essentially one step combination nuclear isolation and DNA staining procedure is used to measure DNA in tissue cells by flow cytometry. Rapid and consistent results are obtained and multiple sampling of the same tissue or comparison between whole tissues and their single cell isolates are also described.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 301,599, filed Sept. 14,1981, now abandoned, which is a continuation-in-part of my earlierapplication Ser. No. 257,155 filed Apr. 24, 1981, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to the preparation of tissues for cytometricanalysis and observation. More particularly a liquid medium is describedfor preparing animal tissues for automated or microscopic analysis inwhich the cell nuclei are separated into discrete, non-agglomeratedunits. The medium and procedure is specifically tailored for flowcytometry.

In order for flow cytometry to be useful in studies of the proliferationkinetics and carcinogenesis of mammalian cells, a consistent, rapidmethod is necessary for the preparation of single cells or nucleisuspensions from solid tissues. Much work has been accomplishedutilizing ascites tumors, cell cultures and hemopoetic tumors. Sincethese samples comprise single cells, preparation for cytometric analysisis simply a matter of staining the cells for DNA content while avoidingcell clumping. Sample preparation of solid tumors or tissues for flowcytometric analysis, however, becomes much more difficult, since thecells must be separated from each other completely in order to minimizefalse DNA histograms generated by two or more cells adhering to oneanother.

There is generally no acceptable procedure for the disaggregation ofcells from tissues. The ideal technique, therefore, must be determinedby trial and error in which the best method of disaggregation is onlyapplicable for a specific tissue in only one species. This problem isamplified in human tumor biopsy analysis. An added problem of scartissue build-up due to either surgical or radiation therapies can causeinconsistent results in multiple biopsies of the same patient.

Several preparative methods have been used for the production of singlecells. The most popular procedures have utilized enzymes with the goalof recovering viable cells which are representative of the whole tissue.Some of the enzymes which have been used include trypsin for murinesquamous-cell carcinoma, pepsin for metastatic human tumors, andcombinations of trypsin-collagenase; see Noel, J. S. et al, "TheDissociation of Transplantable Tumors", J. Histochem Cytochem 25:544(1977). Other methods have applied nonenzymatic, chemical procedures orphysical techniques with the main goal to recover a maximum number ofsingle cells for flow cytometric studies.

In general, these preparative methods are multistep processes whichrequire a customized treatment on each type of tissue under analysis toinsure maximum dissociation. Thus there is the disadvantage that eachprocedure is tissue specific. Also, the reproducibility of the majorityof these methods has not been firmly established. Furthermore, celldissociation is not always complete, and the intepretations of flowcytometric DNA histograms are suspect because of cell aggregationcreating false DNA values. These cell aggregates also make microscopicpattern recognition difficult in that the edge boundaries of aggregatedcells cannot always be clearly defined.

If one is interested in quantitating nuclear DNA in a single cell, thennuclear isolation would be sufficient. The standard biochemicalprocedures for nuclear isolation from tissues usually requirehomogenization and centrifugation steps which have been found to lead toincomplete tissue dissociation, release of DNA from destroyed nuclei andclumping of centrifuged nuclei. These procedures are unacceptable forflow cytometric measurements. Hypotonic solutions, always used withsingle cell preparations but not tissue, without and with the nonionicsurfactant, nonidet P 40 (NP40) have been used to isolate nuclei fromsingle cells for flow cytometric DNA analysis with propidium iodide. Ahypertonic saline solution with NP40 has been utilized to obtain nucleifrom solid tumors; see Vindelov, "Flow Microfluorometric Analysis ofNuclear DNA In Cells From Solid Tumors And Cell Suspension"; VirchowsArch [Cell Pathol] 24:227 (1977).

The objective of the present invention is to provide a dependable, rapidmethod for isolating fluorochrome stained nuclei from normal andcancerous tissues. This one-step procedure of combination nuclearisolation and fluorochrome DNA staining does not require acentrifugation step, therefore, nuclear clumping is avoided. A widerange of tissues may be utilized.

Accordingly it is an object of the present invention to providematerials and methods for measuring DNA in tissue cells by flowcytometry utilizing a one step combination nuclear isolation-DNAfluorochrome staining medium and procedure. The advantages of thisprocedure are that the disclosed nuclear isolation medium (NIM) providesa simple, one step procedure that takes only 5-10 min. to obtain arepresentative sample. This allows for the preparation of multiplesamples from the same tissue in order to define more accurately thecharacteristics of the whole tissue. Moreover, with easily dissociatedtissues, one can compare the single cell and tissue nuclear isolates tosee if their DNA distributions are equivalent. In the majority ofsamples, there is minimal sample debris which results in consistent,high resolution DNA histograms in the 1-2% coefficient of variationrange regardless of the tissue analyzed. Finally, since the NIM iseasily prepared and the disaggregation procedure is a single stepprocess, the whole preparation technique can be standardized, thusallowing accurate comparison of data between laboratories.

Besides the DNA quantitation there are a number of useful parameters inthe cell nucleus to which the present invention is well adapted. Firstof all, nuclear volume may be a useful parameter in characterizing tumordevelopment as shown for erythroleukemic cells. Furthermore, one of themost important criteria for the detection, classification, and stagingof tumor cells is nuclear area. For example, the mean nuclear area forcells in carcinoma in situ is about three times that of normal squamouscell nuclei. Endometrial adenocarcinoma nuclei show an increase in areawhich is directly proportional to the grading scale of these tumors.Also, there is a significant increase in nuclear area in ectocervicalwhen compared to endocervical tumors.

By utilizing high resolution DNA fluorescent measurement with electronicnuclear volume, two parameter scattergrams can be generated in order todevelop a fingerprint technique for the detection and classification oftumors. This correlative analysis would be especially useful in solidtumor studies where single cell preparations are difficult to obtain. Ingeneral, there are a number of antigens, receptors and enzymes that maybe expressed in the nuclei. Certain histocompatability antigens areexpressed in the nuclear membrane. Probably the most important site inthe cell to look for estrogen and progesterone receptors is in thenucleus. Finally, there are several important enzymes in the nucleus.One of these is found in NIM isolated liver nuclei where 5'-ATPaselevels increase significantly in tumor cells. These and otherapplications of the procedures of the present invention will be apparentto those skilled in the art.

DETAILED DESCRIPTION OF THE INVENTION

In its compositional aspect the present invention provides a nuclearisolation medium (sometimes referred to herein as NIM) for separatingcell nuclei that consistently and rapidly allows the isolation of singlecell nuclei by the disaggregation of cells from tissues. The medium isparticularly adapted for use in human tumor biopsy analysis, although itis well suited for other cytological procedures for human and otheranimal tissue.

In order to isolate readily observable and/or analyzable discrete cellnuclei it is necessary to establish a medium that mirrors the externalfluid of the tissue from which the cell nuclei are to be examined, i.e.,the area about the cell in question, and to this end animal-to-animaland tissue-to-tissue variations exist. Thus while my invention will bedescribed essentially with reference to studies of human-derived tissue,it will be understood that through suitable adjustments one may dealwith other animal tissues as well. A medium hospitable to cell nucleishould necessarily have an ionic content comparable to the tonicity ofthe fluid external to the cell, i.e., a tonicity substantially similaror identical the area about the cell in question. Isotonicity is anessential reguirement for the nuclear isolation media of my invention.

There are variations between various classes of animals--as an examplethe average ion content for media for use with human-derived cells is146 mM Na and other ions or a disaccharide may be used. Typically sodiumion is the ion employed. Isotonicity has not been a characteristic ofpreviously described nuclear isolation media for flow cytometricanalysis.

In addition to sodium ion concentration "normal tonicity" is alsorelatable to red blood cells (erythrocytes) of the animal under study.Red blood cells are well recognized as an accurate osmometer. Althoughosmolarity and tonicity are not synonymous, a convenient standard towhich to compare for nuclei are the whole erythrocytes of the animalfrom which the animal tissue sample is isolated.

As used herein the expression "normal electronic cell volume of the cellnuclei", in addition to or as an alternative of sodium ionconcentration, includes a comparison with whole erythrocytes of theanimal in question as a basis for establishing an isotonic solution. Forsuch a comparison, an isotonic medium is established using the nuclearisolation medium as disclosed herein--absent the nonionic surfactantcomponent--and using this solution comparing with the erythrocyte cellvolume when these cells are suspended in the animal's natural peripheralblood fluid. When observed via electronic means the two electronicerythrocyte cell volume distributions appear substantially completelyidentical to the extent that the coefficient of variation (CV) betweenthe two, expressed as a percentage and peak channel number, are both+2%. These measurements are described in more detail in Thornthwaite etal, "The Use of Electronic Cell Volume Analysis . . . ", ScanningElectron Microscopy (1978), Volume II, pp. 1123-1130, the disclosure ofwhich is incorporated by reference.

A second essential ingredient of the nuclear isolation media is a minoramount of a nonionic surfactant. Suitable nonionic surfactants aredescribed in McCutcheon's Detergents and Emulsifiers (1977). Preferredare Nonidet P 40, an octylphenol ethylene oxide condensate with anaverage of 9 moles of ethylene oxide available as a 27% solution fromAccurate Scientific and Chemical Co., Triton X-100, an octylphenoxypolyethoxy ethanol available from Rohm & Haas, and Octy Glycoside, a1-0-n-octyl-beta-d-glycopyranoside available from Sigma Chemical Co. Asuitable surfactant is easily determined by empirical selection andevaluation.

The amount of surfactant included in the media should be sufficient thatdissociation is nearly complete, that is there is less than 2% clumpednuclei when observed under a microscope, substantial numbers of clumpedcell nuclei evidence incomplete cell dissolution. The desirable level isan amount that allows a smooth and even flow in DNA flow cytometricanalysis equipment, as described more fully below. Excessive amounts ofsurfactant are evidenced by loss of cell integrity and swollen orshrunken cells. Surfactant quantity is also indicated by the intensityof the fluorescent stain when such stain is used. Excessive amounts ofsurfactant decreases the fluorescence of the stain to an unacceptablelevel.

As indicated, the preferred surfactant is Nonidet P 40 (available as a27% by weight solution) which is included in the media within the rangeof about 0.1 to about 5% v/v and preferably in the range of about 0.6 toabout 2% v/v.

Also a microscopy-type stain is usually included in the medium.Illustrative stains include protein stains, such as fluoresceinisothiocyanate; RNA stains such as acridine orange; antibody stains;receptor binding compounds; and enzyme substrate stains. Preferred inflow cytometric analysis are the DNA fluorochrome-type stains such as4',6-diamidino-2-phenylindole-2 HCl and propidium iodide. DNA stains forfluorescence and/or phosphorescence may also be used. The stain isincluded in an amount as is customarily used in cytometric procedures.Combinations of various stains may also be used.

An environment hospitable to the cell nuclei preferably also includesserum albumin in an amount selected to match that of the physiologicalstate of the animal from which the tissue was taken. When used serumalbumin is present in an amount of about 0.05 to about 1.0% andpreferably in an amount of about 0.2%. Divalent cations, particularlycalcium and magnesium, are included in the media as such ions have beenfound important in the preservation of the nuclear membrane; see R.Hancock, J. Cell Physiol 97:37 (1978).

The media should have a pH of about 5 to about 8 and preferably fromabout pH 6.5 to about pH 7.5. The specific pH will be selected to beequivalent to the physiological pH of the animal in question. A bufferor buffer system is also included in an optional ingredient to maintainthe pH of the media.

In its procedural aspect the invention provides a convenient one-stepmethod of preparing cells and tissue samples for cell nuclei observationand analysis using flow or static cytometric measurements. Maximum celldissociation is achieved and cell aggregation is avoided in thisprocedure. A wide variety of tissues may be used, including frozentissue, which when subjected to nuclear volume and DNA fluorescence flowcytometric analysis provides a marker for neoplastic cells.

A typical procedure includes placing 0.2 g of tissue under study in apetri dish, adding 3 ml. of the nuclear isolation media, describedabove, mincing the tissue with a sharp instrument and waiting 5 minutes.The tissue and liquid mixture are filtered through a 70 micron filter toremove cell debris. The nuclei, usually 10 to 15 microns in diameter,pass the filter and are collected in a vessel together with the passedliquid. Optionally the cells/liquid mixture is syringed or subjected toultrasonication to assure single cell nuclei. The resulting preparationis complete and ready for static or flow cytometric analysis. It will beunderstood that this is but one of many dissociation procedures that maybe employed; the skilled analytical worker will have no difficulty inachieving the desired degree of dissociation following the proceduresherein described.

The invention will now be described with reference to the followingexample, considered illustrative but not limiting, the invention.

Tissue Sources:

A variety of tissues was used in this study. Sarcoma tumors wereproduced by 3-methylcholanthrene (MCA) induction in C57BL/6J, male mice.Their associated metastatic nodules were isolated from the lungs of micewho had their intramuscular induced tumor-bearing legs amputated. Thisdecrease in tumor burden promoted metastatic growth. The followingnormal tissues were utilized from the C57BL/6J mice: liver, bone marrowand testicle. A murine brain tumor was prepared for flow cytometricanalysis. Wistar rats were utilized as the source of pancreatic islets.Normal human samples included peripheral blood leukocytes and colonmucosa. Human tissue samples from biopsy specimens included colon,sarcoma and brain tumors.

Nuclear Isolation and Staining Technique:

Nuclear isolation and DNA fluorochrome staining were combined in arapid, one step procedure. The nuclear isolation medium (NIM) consistedof 0.01 M phophate buffered isotonic saline (146 mM) with calcium (1.0mM CaCl₂) and magnesium (0.5 mM MgSO₄.7 H₂ O) which contained 0.6% NP40(v/v) (Accurate Scientific and Chemical Co., Hicksville, N.Y.) and 0.2%bovine serum albumin (BSA) (w/v) (Fraction Five GIBCO). This formulationwas developed after a series of studies with various physiologic buffers(pH 6.5-7.5) and different concentrations of the various components. TheBSA was not an absolute requirement but did help stabilize the nuclearmembranes. A commercially available phosphate buffer (0.01 M) saline(152 mM) with calcium and magnesium (Dulbecco, GIBCO) was similar in theNIM phosphate buffered saline solution and substituted very well for theabove formulation.

The DNA fluorochrome 4'6-diamidino-2 phenylindole-2 HCl (DAPI) (AccurateScientific and Chemical Co.) was dissolved in the nuclear isolationmedium at a concentration of 10 g/ml. Tissues (0.1-0.3 g) were mincedwith scalpels in the NIM-DAPI stain solution (0.1 g/ml) for about 1-2min. After mincing, the tissues were left in the NIM-DAPI for 3 min. atroom temperature. The suspensions were then filtered through a 70- nylonmesh to remove tissue debris. The nuclei were syringed through a26-gauge needle back and forth twice and filtered through the 70- mesh.This syringing procedure was used with all of the samples only as aprecautionary measure to insure single nuclei, since the NIM stepresulted in almost complete nuclear dissociation. The stained nuclearisolates were observed using a fluorescent microscope to determine thepercentage of nuclear aggregates. If greater than 2% of the nuclei werejoined together, they were syringed again. Samples not analyzedimmediately were left at 4° C. for up to 48 hr. and then syringed andfiltered before analysis. The dissociative activity of the NIM wasstable for about 3 months if it was kept in the refrigerator.

Flow Cytometry:

An ICP-22 Flow Cytometer (Ortho Instruments, Westwood, Mass.) wasinterfaced to a SUE Lockheed computer (Lockheed Electronics, Plainfield,NJ). With this system, the measurement, acquisition and analysis of theDNA content of isolated nuclei was accomplished. DAPI fluorescence wasquantitated utilizing a BG 1 exciter filter, a TK 405 dichroic and LP395 barrier filter. The nuclei were analyzed at a rate of 300-400nuclei/sec. In cases where the Coulter Epics-IV cell sorter (CoulterElectronics, Hialeah, FL) was utilized, propidium iodide (Calbiochem)was substituted for DAPI in the NIM at a concentration of 50 g/ml.

Fluorescence Photomicroscopy:

A Zeiss Photomicroscope III with a 100 watt mercury lamp was used inphotographing the DAPI stained nuclei. A BG 3 exciter filter was usedwith an additional cut-off filter to isolate the mercury line rangearound 405 nm. The blue-green fluorescence was isolated utilizing adichroic beam splitter and LP 475 barrier filter.

For cell populations which can be teased into single cell suspensions,for example, bone marrow, lymphoid organs and peripheral blood, themedia and technique of the present invention is not necessary if a DNAspecific stain, such as DAPI, is utilized. NIM is required in caseswhere one utilizes an intercalating dye, such as propidium iodide whichwill bind with RNA as well as DNA. By isolating nuclei, the staining bycytoplasmic RNA is eliminated.

The procedures and media of this invention are useful not only inpreparing tissues for flow cytometric analysis, for instance indetermining S phase DNA synthesis, but also in tumor detection andradiation or chemotherapy monitoring. A detailed discussion of specificadaptations for flow analysis, observations and other evidence ofperformance is contained in Thornthwaite et al, "Preparation of Tissuesfor DNA Flow Cytometric Analysis", Cytometry, Volume 1, No. 3, pp.229-237 (1980) the disclosure of which is hereby incorporated byreference. The technique of and equipment for flow analysis is describedin the text Flow Cytometry and Sorting, Melamed, et al, editors, JohnWiley & Sons (1979).

Another use for the media of the present invention is to remove intactnuclei from a tissue culture plate. Previous procedures employed variousenzyme-containing solutions which released the cells from the tissueculture plate and also altered the condition of the nuclei so released.Using the nuclear isolation media described above the cell nuclei cameloose from the tissue culture plate in a single step.

The tissue sources are maintained at about room temperature or frozentissues, i.e. -80° C., may be conveniently used.

The nuclear isolation technique is useful to compare normal cell nucleiwith suspected cell nuclei as in DNA flow cytometric analysis in orderto establish "normal" amounts of DNA in normal tissue and thus comparethis with suspected tissue of the same histoloqical type. Priorprocedures required a standardization step with "normal" single cells,such as white blood cells. This was then compared with the cell nucleiof the suspected tissue, which in all cases except white blood cells wasof a different tissue type. Using the procedure of the invention it isnow possible to dissociate nuclei of the same histological type tissuefor both normal and "abnormal" tissue. Thus a bank of stock tissues maybe retained, and frozen, i.e. about -80° C. if desired, and madeavailable to compare and cross-analyze with suspected cells of the samehistological tissue type.

The nuclear isolation media of the present invention is capable oflysing red blood cells. This is a useful property, particularly whenpreparing whole blood for analytical cytology. The nuclear isolationmedia lyses and helps to remove red cells, which contain no DNA, leavingthe white cell DNA nuceli intact for study and analysis in a singlestep. Previous procedures required an enzymatic red cell lysis step.

What is claimed is:
 1. A nuclear isolation medium for DNA flowcytometric analysis used to dissociate an animal tissue sample intodiscrete, non-agglomerated cell nuclei, said medium consistingessentially ofa nonionic surfactant present in an amount sufficient toseparate the cell nuclei while maintaining nuclear membrane integrity; amicroscopy stain for staining the DNA cell content; serum albumin in anamount substantially the same as the physiological state of the tissuesample; and divalent cations in an amount substantially equal to theamounts of said cations in the fluid surrounding the nuclear membrane ofthe tissue sample, dissolved in an aqueous solution that issubstantially completely isotonic with and maintains the normalelectronic volume of the cell nuclei, the medium having a pH that is inthe range of pH 6.5 to 7.5 and is substantially identical to thephysiological pH of the animal from which the tissue originates.
 2. Thenuclear isolation medium of claim 1 wherein the divalent cations aremagnesium and/or calcium and the serum albumin concentration is about0.05% to 1.0% v/v.
 3. The nuclear isolation medium of claim 1 whereinthe sodium ion concentration is about 146 mM Na⁺.
 4. Discrete,non-agglomerated cell nuclei contained in the nuclear isolation mediumof claim
 1. 5. A method of dissociating cell nuclei from an animaltissue containing same into discrete, nonagglomerated individual cellnuclei, said method comprising the steps of:(a) placing a tissue samplein a nuclear isolation medium containing an isotonic solution of acell-separating amount of a nonionic surfactant and at least onemicroscopy stain; (b) physically mincing the tissue sample while incontact with the nuclear isolation medium; (c) allowing the mincedtissue sample to remain in contact with the nuclear isolation mediumuntil a quantity of single nuclei have been separated; and (d) removingthe single nuclei from the remaining tissue and cellular debris.
 6. Thecell nuclei dissociation method of claim 5 wherein the nuclear isolationmedium is buffered.
 7. The cell nuclei dissociation method of claim 5wherein the nuclear isolation medium also contains serum albumin in anamount substantially the same as the physiological state of the animalfrom which the tissue was taken.
 8. The cell nuclei dissociation methodof claim 5 wherein the serum albumin is present in an amount of fromabout 0.05% to about 1.0% by weight.
 9. The cell nuclei dissociationmethod of claim 5 wherein the nuclear isolation medium also containsdivalent cations in an amount substantially equal to the amounts of saidcations in the fluid surrounding the nuclear membrane of the tissuesample.
 10. The cell nuclei dissociation method of claim 5 wherein thedivalent cations are calcium, magnesium or their mixtures.
 11. The cellnuclei dissociation method of claim 9 wherein the nuclear isolationmedium also contains:serum albumin in an amount substantially the sameas the physiological state of the animal from which the tissue wastaken, and magnesium plus calcium ions in amounts substantially equal tothe amounts of said cations in the fluid surrounding the nuclearmembrane of the tissue sample.
 12. The cell nuclei dissociation methodof claim 6 wherein the single cell nuclei are removed from the tissueand cellular debris in step (d) by filtration.
 13. The cell nucleidissociation method of claim 5 including the additional step of:(e)subjecting the separated nuclei to DNA flow cytometric analysis.
 14. Amethod of preparing tissue for DNA flow cytometric analysis bydissociating cell nuclei from an animal tissue containing same intodiscrete, non-agglomerated individual cell nuclei, said methodcomprising the steps of:(a) placing a tissue sample in a nuclearisolation medium containing a solution of a cell-separating amount of anonionic surfactant and a DNA stain in a buffered aqueous solution thatis substantially isotonic with and maintains the normal electronicvolume of the cell nuclei, said medium having a pH in the range of 5 to8 and substantially the same as the physiological pH of the animal fromwhich the tissue originates; (b) physically mincing the tissue samplewhile in contact with the nuclear isolation medium; (c) allowing theminced tissue sample to remain in contact with the nuclear isolationmedium until at least an analyzable quantity of single nuclei have beenseparated; (d) filtering and removing the single nuclei from the tissueand cellular debris, and (e) subjecting the thus separated nuclei to DNAflow cytometric analysis.
 15. The method of claim 14 wherein the nuclearisolation medium also contains serum albumin in an amount substantiallythe same as the physiological state of the animal from which the tissuewas taken.
 16. The method of claim 15 wherein the nuclear isolationmedium additionally contains magnesium and/or calcium ions in an amountsubstantially equal to the amounts of said ions in the fluid surroundingthe nuclear membrane of the tissue sample.
 17. The method of comparingnormal cell nuclei with suspended cell nuclei both said nuclei of thesame histological type, said method comprising the steps of:(1) placinga normal tissue sample in a nuclear isolation medium composed of a cellnucleus-separating amount of a nonionic surfactant and a fluorescent,phosphorescent, or absorbance stain, the surfactant and stain containedin a buffered aqueous solution that is substantially isotonic with thecell nuclei and has a pH (a) in the range of 5 to 8, and (b) issubstantially the same as the physiological pH of the animal from whichthe tissue was taken,and separating a first group of single nuclei fromthe remaining normal tissue and cellular debris; (2) placing a suspectedtissue sample in said nuclear isolation medium and separating a secondgroup of single nuclei from the remaining suspected tissue and cellulardebris; and (3) comparing the first group of single nuclei with thesecond group of nuclei.
 18. The comparison method of claim 17 whereinthe groups of cell nuclei are compared by analytical cytology analysisin step (3).
 19. The comparison method of claim 17 wherein the nuclearisolation medium also contains serum albumin in an amount substantiallythe same as the physiological state of the animal from which the sampleswere taken.
 20. The method of claim 19 wherein the nuclear isolationmedium additionally contains magnesium and/or calcium ions in an amountsubstantially equal to the amounts of said ions in the fluid surroundingthe nuclear membrane of the tissue sample.
 21. The comparison method ofclaim 17 wherein the normal tissue sample is taken from one individualand the suspected tissue sample is taken from a second individual.